| Wheat (Triticum aestuvum L.) is a staple food crop in the world, butdrought seriously limits the yield of wheat. It is one of the economical andeffectual approachs to improve and utilize the drought-tolerant potential inwheat. Cloning key genes involved in drought tolerance in plants is ofsignificance on improving the drought tolerance of crops via geneticengineering. Developing the molecular marker of functional genes willaccelerate the molecular marker selection in wheat. In addition, singlenucleotide polymorphism (SNP) is hitherto the most abundant markers inwheat. Consequently, developing SNP marker of functional genes possessescrucial academic and practical significance.During their evolution, plants have acquired adaptive strategies, such asregulating respiration metabolism, to cope with various stress conditions. Themitochondrial respiratory chain is vital to respiration metabolism. CoenzymeQ (CoQ/Q), a member of the mitochondrial respiratory chain, is present in allcells and membranes and has also several other functions of great importancefor the cellular metabolism. CoQ is an electron carrier in the mitochondrialrespiratory chain and required for bc1 complex stability. Additional functionsof CoQ include its action as a lipid soluble anti-oxidant, and its requirementfor the regulation of aerobic and anaerobic metabolism through theArcA/ArcB signal transduction system in Escherichia coli. ABC1, which isidentical to CoQ8, was originally isolated as a multicopy suppressor of acytochrome b mRNA translation defect, and it can stabilize bc1 complex ofthe electron transport chain in the mitochondria. It was presumed that ABC1could be a novel chaperonin. ABC1 protein in yeast has a nuclear ormitochondrial subcellular location. Abc1/coq8 null mutants leaded to CoQbiosynthesis defective and respiratory deficiency in yeast. UbiB, oneprokaryotic homolog of ABC1, is required for the first monooxygenase stepin Q-biosynthesis in E. coli. ABC1 in human affected the respiratory chainand was involved in apoptosis through the mitochondrial pathway by way ofcooperation with other mitochondrial proteins. Furthermore, ABC1 plays the important role of the complexⅡ,ⅢandⅣin the respiration chain.However, the function of ABC1 is less reported in plants.The objective of this research was to isolate the full-length sequence ofABC1 gene from hexaploid wheat; to analyze its expression character invarious stress conditions; to develop markers of TaABC1 gene based on theSNP in the sequence, and mapping it on the genetic linkage maps.The results are as following:(1) We cloned TaABC1L, an abiotic stress-responsive gene, fromcommon wheat with a combination of reverse Northern blot screening,bioinformatics and RT-PCR strategies. A candidate EST, which was allup-regulated at 1 h, 6 h and 12 h water stress, was selected from the cDNAlibrary constructed with mRNA isolated from the 2-leaf seedling of commonwheat Hanxuan 10 treated with 1 h water stress, by reverse Northem blotting.The EST was extended by in silico cloning via eight high homologous wheatESTs (Accession number: BQ802859, BQ842082, BF200834, CD874213,CD874214, CD883956, CV760115, CD918331) from EST database at NCBI.By designing primers based on the extended sequence, the full-length cDNAof TaABC1L was amplified via RT-PCR using primers TaABC1L-F/R. Thefull-length cDNA sequence was predicted to contain a 1434bp open readingframe, and to encode a polypeptide of 477 amino acids with a calculatedmolecular mass of 54.55 kDa and isoelectric point (pI) of 8.34. Sequenceanalysis indicated this putative protein includes ABC1 characteristic domainarchitecture (123～243aa) and AARF domain (42～369aa), but ABC1precursor mitochondrion transit peptide (PD017350) was not observed.Therefore the gene was designated TaABC1L (TaABC1-like). The alignmentof the putative amino acid sequence of the wheat ABC1L gene and relatedsequences from public databases was performed. The result revealed thatTaABC1L was 88%, 62%, 31%, 29%, 28% and 29% identical to ABC1Lfrom Oryza sativa, unknown protein from Arabidopsis thaliana, ABC1 fromTetrahymena thermophila, ADCK1 from Homo sapiens, Gallus gallus andMus musculus, respectively. The expression pattern of TaABC1L at transcription level was investigated by real-time quantitative RT-PCR, whichrevealed that TaABC1L was distinctly responsive to hyperosmolality(-0.5MPa, PEG-6000), high salinity (250 mmol L-1 NaCl), low temperature(4℃) stresses and abscisic acid (50μmol L-1 ABA) treatment. Expressionalpeaks of TaABC1L responsed to PEG, NaCl and 4℃stresses appeared at 24 h,1 h and 48 h, with 13.9-fold, 30-fold and 9.8-fold as high as that of thecontrol, respectively. However, the expressional intensity of the peak with 12h ABA treatment was only 2-fold as high as that of the control. Our studiesstrongly suggested that TaABC1L could respond to environmental stressesincluding salinity, low temperature, osmotic stresses and exogenous ABAtreatment.(2) Using the primer pair P1/P3, ABC1L genes were amplified fromhexaploid wheat W7984, Opata85 and their wild relative species. Partialgenome sequences of TaABC1L from hexaploid wheat and related diploidspecies were analyzed simultaneously. A and D genome sequences fromhexaploid wheat were unambiguously related to a diploid sequence, while Bgenome sequences from W7984 and Opata85 clustered in the same groupwere not closer to Ae. speltoides sequence. Based on the similarity, we easilydesigned A-genome specific primer pair P4/P5, and D-genome specificprimer P7. B-genome specific primer P6 was still devised according to thesequence of Ae. speltoides, although actual B-genome of common wheat isnot closer to Ae. speltoides. P4/P5, P6/P3 and P7/P3, three genome-specificprimer pairs for TaABC1L, were tested on T. urartu, Ae. speltoides, Ae.tauschii, T. turgidum, T. aestivum W7984 and Opata85. Specificamplification products, as expected, were obtained for each genome-specificprimer pair. TaABC1L gene was further assigned to chromosomes 3A, 3B and3D nulli-tetrasomic lines of Chinese Spring, respectively.(3) The CAPS marker was developed based on A- and B-genome SNP inTaABC1L. Allele variations were observed in predicted restriction enzymetarget sites within TaABC1L-A gene and TaABC1L-B gene. Two haplotypesfor TaABC1L-A gene contain a canonical MseI site, which has a SNP at the second base. The corresponding structures for the TaABC1L-B gene contain acanonical SalI site, in which a SNP was detected at the first base locus.Amplicon-specific PCR was performed for TaABC1L-A or TaABC1L-B on arecombination inbred lines (RIL) derived from the cross between W7984 andOpata85. Following digestion with the diagnostic restriction enzyme, twogenotypic classes were revealed for TaABC1L-A: a class with the two bandsresembling the genotype of parental W7984 and a class with one bandresembling the genotype of Opata85. The pattern with the diagnosticrestriction enzyme for TaABC1L-B revealed that there was only a class withone band in both W7984 and Opata85 parental genotypes, while the size ofthe band was bigger in W7984 parental genotype than that of Opata85parental genotype. The Xabc11-A CAPS marker was located, with a highdegree of confidence (LOD＞4.0), within the framework of the referencegenetic map in the 41.7 cM interval between the flanking markers Xmwg30and Xmwg570 on chromosome 3A, with a most probable location adjacent tothe Xmwg30 locus. The Xabc11-B CAPS marker was located with a highdegree of confidence in the 29.2 cM interval between XATPase.2 andXbcd147 on chromosome 3B, with a most probable location adjacent to theXbcd147 locus.(4) The AS-PCR marker was developed based on D-genome SNP inTaABC1L. D-genome allele-specific primer, P8 was used to amplify theW7984 allele of TaABC1L with SNP in the 3'-terminal (W7984=C, Opata85=T). A mismatch base, T was introduced at the second site of 3'-terminal inthe allele-specific primer in order to enhance specific amplification. TheP7/P8 primer pair was tested on the RIL. The banding patterns showed that aclass resembling W7984 parental genotype presented one specific-band witha size of 729bp, while Opata85 parental genotype was absent on the sameposition. In addition, one band shared by parent paire was concomitant. TheAS-PCR marker that we named Xabc11-D was then located on chromosome3D, 1.3 cM and 2.3 cM from the Xfba330 and Xgwm645, respectively. |
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